Electrophoresis

5 min read Updated Apr 18, 2026

Electrophoresis separates charged molecules by driving them through a gel under an electric field. Small or highly charged molecules move fast. Large or weakly charged molecules move slowly. In protein biochemistry, electrophoresis is the standard way to check how pure a preparation is and to estimate molecular weights.

SDS-PAGE - Size Only

SDS-PAGE is the gold-standard technique for separating proteins by size. SDS (sodium dodecyl sulfate) is a detergent that does three things:

Coats every protein with many SDS molecules, giving all proteins a uniformly strongly negative charge proportional to length.
Denatures (unfolds) the proteins into extended rods.
Disrupts non-covalent interactions.

Beta-mercaptoethanol (or dithiothreitol) is added to reduce disulfide bonds, so multi-chain proteins are separated into individual subunits.

After SDS treatment, every protein has roughly the same charge-to-mass ratio. In the polyacrylamide gel, separation depends only on size: small proteins move quickly through the gel pores, large proteins move slowly.

An SDS-PAGE polyacrylamide gel after staining, showing a molecular weight ladder lane and several sample lanes with bands at different positions corresponding to protein sizes — SDS-PAGE gel. Each lane contains a sample; each band represents a different-sized protein. The left lane typically has a ladder of known molecular weight markers for sizing. *Credit: Wikimedia Commons, CC BY-SA*

Native PAGE - No SDS

In native PAGE, you leave the protein folded and natively charged. Separation depends on a combination of size, shape, and net charge. Native PAGE is useful when you want to preserve biological activity (a protein that is still functional after the gel). It gives less clean size information than SDS-PAGE because shape and charge vary among proteins.

Isoelectric Focusing (IEF) - Charge Only

A gel is cast with a pH gradient. When an electric field is applied, a protein migrates toward the pole opposite its net charge. As it moves, it crosses pH zones that change its protonation state. When it reaches the pH that equals its isoelectric point (pI), the protein has net zero charge and stops moving. Each protein “focuses” at its own pI.

Isoelectric focusing process. A pH gradient in the gel causes proteins to migrate until each reaches the pH matching its isoelectric point, where it has zero net charge and stops moving — Isoelectric focusing. Each protein stops migrating when the local pH equals its pI. Sharp bands correspond to individual pI values. *Credit: Wikimedia Commons, CC BY-SA*

IEF is often used to detect small charge differences between protein variants - for example, distinguishing isoforms that differ by a single amino acid charge or by a post-translational modification.

2D Gel Electrophoresis - Charge Then Size

Combining IEF and SDS-PAGE gives 2D gel electrophoresis, the highest-resolution method for separating complex protein mixtures.

Run IEF in one dimension (horizontal), separating by pI.
Rotate the strip 90 degrees, apply SDS, and run SDS-PAGE in the perpendicular dimension (vertical), separating by size.

The result is a 2D “spot pattern” where each protein appears as a single dot at unique coordinates of (pI, molecular weight). Thousands of proteins can be resolved in one gel, and the pattern can be compared between healthy and diseased tissue to find differentially expressed proteins.

Detecting the Bands

After the gel is run, you need to visualize the proteins:

Coomassie blue or silver stain binds proteins nonspecifically and shows all bands.
Western blot: transfer gel contents to a membrane, then probe with antibodies to detect one specific protein. This is the standard technique for proving a specific protein is present.

What does SDS do to proteins before electrophoresis?

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SDS is a detergent that denatures proteins (unfolds them), coats them in negative charge proportional to length, and disrupts non-covalent interactions. The result is that every protein has a similar charge-to-mass ratio, so migration depends only on size. Without SDS, migration would depend on size, shape, and native charge all at once.

In isoelectric focusing, what determines where a protein stops moving?

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The protein stops migrating when the local pH of the gel equals the protein's isoelectric point (pI). At that pH, the protein has zero net charge, so the electric field no longer drives it forward. Different proteins stop at different pI values, producing sharp bands along the pH gradient.

Why would a researcher use a 2D gel instead of a standard SDS-PAGE?

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SDS-PAGE separates proteins by size only. If two proteins have very similar molecular weights but different pI values, they appear as a single band on SDS-PAGE. 2D gels add an IEF dimension, separating those same two proteins into distinct spots. This is how 2D gels resolve thousands of proteins in a complex lysate.